Method of evaluating patient hemostasis

ABSTRACT

A hemostasis analyzer, such as the Thrombelastograph® (TEG®) hemostasis analyzer is utilized to measure continuously in real time, the hemostasis process from the initial fibrin formation, through platelet-fibrin interaction and lysis to generate blood hemostasis parameters. The measured blood hemostasis parameters permit evaluation of a patient hemostasis condition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/384,345 filed Mar. 7, 2003 entitled Protocol for MonitoringPlatelet Inhibition now U.S. Pat. No. 7,179,652, which is acontinuation-in-part of U.S. patent application Ser. No. 09/591,371filed Jun. 9, 2000 entitled Method and Apparatus for MonitoringAnti-Platelet Agents now U.S. Pat. No. 6,613,573, which is acontinuation-in-part of U.S. patent applications Ser. No. 09/255,099,filed Feb. 22, 1999 entitled Method and Apparatus for MeasuringHemostasis, now U.S. Pat. No. 6,225,126, the disclosures of which arehereby expressly incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a protocols for monitoring patienthemostasis and in particular protocols for monitoring the efficacy ofpatient hemostasis therapies.

BACKGROUND OF INVENTION

Blood is the circulating tissue of an organism that carries oxygen andnutritive materials to the tissues and removes carbon dioxide andvarious metabolic products for excretion. Whole blood consists of a paleyellow or gray yellow fluid, plasma, in which are suspended red bloodcells, white blood cells, and platelets.

An accurate measurement of hemostasis, i.e., the ability of a patient'sblood to coagulate and dissolve, in a timely and effective fashion iscrucial to certain surgical and medical procedures. Accelerated (rapid)and accurate detection of abnormal hemostasis is also of particularimportance in respect of appropriate treatment to be given to patientssuffering from hemostasis disorders and to whom it may be necessary toadminister anti-coagulants, antifibrinolytic agents, thrombolyticagents, anti-platelet agents, or blood components in a quantity whichmust clearly be determined after taking into account the abnormalcomponents, cells or “factors” of the patient's blood which may becontributing to the hemostasis disorder.

Hemostasis is a dynamic, extremely complex process involving manyinteracting factors, which include coagulation and fibrinolyticproteins, activators, inhibitors and cellular elements, such as plateletcytoskeleton, platelet cytoplasmic granules and platelet cell surfaces.As a result, during activation, no factor remains static or works inisolation. Thus, to be complete, it is necessary to measure continuouslyall phases of patient hemostasis as a net product of whole bloodcomponents in a non-isolated, or static fashion. To give an example ofthe consequences of the measuring of an isolated part of hemostasis,assume that a patient developed fibrinolysis, which is caused by theactivation of plasminogen into plasmin, an enzyme that breaks down theclot. In this scenario, a byproduct of this process of fibrinogendegrading product (FDP), which behaves as an anticoagulant. If thepatient is tested only for anticoagulation and is treated accordingly,this patient may remain at risk due to not being treated withantifibrinolytic agents.

The end result of the hemostasis process is a three-dimensional networkof polymerized fibrin(ogen) fibers which together with plateletglycoprotein IIb/IIIa (GPIIb/IIIa) receptor bonding forms the final clot(FIG. 1). A unique property of this network structure is that it behavesas a rigid elastic solid, capable of resisting deforming shear stress ofthe circulating blood. The strength of the final clot to resistdeforming shear stress is determined by the structure and density of thefibrin fiber network and by the forces exerted by the participatingplatelets.

Platelets have been shown to effect the mechanical strength of fibrin inat least two ways. First, by acting as node branching points, theysignificantly enhance fibrin structure rigidity. Secondly, by exerting a“tugging” force on fibers, by the contractability of plateletactomyosin, a muscle protein that is a part of a cytoskeleton-mediatedcontractability apparatus. The force of this contractability furtherenhances the strength of the fibrin structure. The platelet receptorGPIIb/IIIa appears crucial in anchoring polymerizing fibers to theunderlying cytoskeleton contractile apparatus in activated platelets,thereby mediating the transfer of mechanical force.

Thus, the clot that develops and adheres to the damaged vascular systemas a result of activated hemostasis and resists the deforming shearstress of the circulating blood is, in essence a mechanical device,formed to provide a “temporary stopper,” that resists the shear force ofcirculating blood during vascular recovery. The kinetics, strength, andstability of the clot, that is its physical property to resist thedeforming shear force of the circulating blood, determine its capacityto do the work of hemostasis, which is to stop hemorrhage withoutpermitting inappropriate thrombosis. This is exactly what theThrombelastograph® (TEG®) system, described below, was designed tomeasure, which is the time it takes for initial fibrin formation, thetime it takes for the clot to reach its maximum strength, the actualmaximum strength, and the clot's stability.

Hemostasis analyzer instruments have been known since Professor HelmutHartert developed such a device in Germany in the 1940's. One type ofhemostasis analyzer is described in commonly assigned U.S. Pat. No.5,223,227, the disclosure of which is hereby expressly incorporatedherein by reference. This instrument, the TEG® hemostasis analyzer,monitors the elastic properties of blood as it is induced to clot undera low shear environment resembling sluggish venous blood flow. Thepatterns of changes in shear elasticity of the developing clot enablethe determination of the kinetics of clot formation, as well as thestrength and stability of the formed clot; in short, the mechanicalproperties of the developing clot. As described above, the kinetics,strength and stability of the clot provides information about theability of the clot to perform “mechanical work,” i.e., resisting thedeforming shear stress of the circulating blood; in essence, the clot isthe elementary machine of hemostasis, and the TEG® analyzer measures theability of the clot to perform mechanical work throughout its structuraldevelopment. The TEG® system measures continuously all phases of patienthemostasis as a net product of whole blood components in a non-isolated,or static fashion from the time of test initiation until initial fibrinformation, through clot rate strengthening and ultimately clot strengththrough fibrin platelet bonding via platelet GPIIb/IIIa receptors andclot lysis.

Platelets play a critical role in mediating ischemic complications inprothrombotic (thrombophilic) patients. The use of GPIIb/IIIa inhibitoragents in thrombophilic patients or as an adjunct to percutaneouscoronary angioplasty (PTCA) is rapidly becoming the standard of care.Inhibition of the GPIIb/IIIa receptor is an extremely potent form ofantiplatelet therapy that can result in reduction of risk of death andmyocardial infarction, but can also result in a dramatic risk ofhemorrhage. The reason for the potential of bleeding or non-attainmentof adequate therapeutic level of platelet inhibition is theweight-adjusted platelet blocker treatment algorithm that is used inspite of the fact that there is considerable person-to-personvariability. This is an issue in part due to differences in plateletcount and variability in the number of GPIIb/IIIa receptors per plateletand their ligand binding functions.

Since the clinical introduction of the murine/human chimeric antibodyfragment c7E3 Fab (abciximab, ReoPro®), several synthetic forms ofGPIIb/IIIa antagonists have also been approved such as Aggrastat®(tirofiban) and Integrilin® (eptifibatide), resulting in widespread andincreasing use of GPIIb/IIIa inhibitor therapy in interventionalcardiology procedures.

Before the introduction of the method and apparatus described in theafore-mentioned U.S. Pat. No. 6,613,573 there was no rapid, reliable,quantitative, point-of-care test for monitoring therapeutic plateletblockade. Although the turbidimetric aggregation test has been used tomeasure the degree of platelet GPIIb/IIIa receptor blockade in smallclinical studies and dose-finding studies, its routine clinical use fordosing GPIIb/IIIa receptor antagonists in individual patients has notbeen feasible. Aggregation is time-consuming (more than one hour),expensive to run, requires specialized personnel for its performance,and is not readily available around the clock; therefore it cannot beemployed at the point-of-care for routine patient monitoring and doseindividualization. To be clinically useful, an assay of plateletinhibition must provide rapid and reliable information regardingreceptor blockade at the bedside, thereby permitting dose modificationto achieve the desired anti-platelet effect.

The turbidimetric aggregation test is based on the photometricprinciple, which monitors the change in the specimen's optical density.Initially, a minimal amount of light passes through the specimen, asfunctional platelets are activated by the turbidimetric test, plateletaggregation occurs via platelet GPIIb/IIIa receptor and fibrin(ogen)bonding as illustrated in FIG. 1, and thus light transmission increases.When platelets are inhibited through GPIIb/IIIa receptor blockade, lighttransmission increases proportionally.

Another commercially available system measures fibrinogen-plateletbonding using beads coated with a fixed amount of an outside source of“normal” fibrinogen. Therefore, this system uses a non-patient source of“normal” fibrinogen and cannot detect a patient in a prothrombotic state(hypercoagulable) due to a higher patient level of fibrinogen, or detecta hemorrhagic state (hypocoagulability) due to a low patient level offibrinogen. Additionally, this system shows only bonding withoutdetection of the breakdown of that bonding. Therefore, in the presenceof thrombolysis, the assessment of platelet GPIIb/IIIa receptor blockadeby the system may not be accurate.

Fibrinogen-platelet GPIIb/IIIa bonding is the initial phase of plateletaggregation, or a primary hemostasis platelet plug, which goes on toform the final fibrin-platelet bonding. Thus it is not sufficient tomeasure only the initial stage of fibrinogen-platelet bonding, which maynot accurately reflect final fibrin-platelet bonding via the GPIIb/IIIareceptor. While the turbidimetric and other photometric systems dodetect initiation of platelet aggregation via fibrinogen-plateletGPIIb/IIIa receptor bonding, it may not accurately reflect finalfibrin-platelet bonding via the GPIIb/IIIa receptor.

Significant among the limitations of systems that use beads coated with“normal” fibrinogen is that this “normal” fibrinogen may not reflecteither the quantity or the functionality of a specific patient's ownfibrinogen. Therefore, fibrinogen-platelet GPIIb/IIIa receptor blockadeas measured by such systems is but a rough estimate of the patient'sindividual fibrinogen-platelet GPIIb/IIIa blockade of the initial phaseof platelet aggregation.

This is a significant limitation in certain high risk patient subgroups,which may need treatment with a platelet inhibition agent, may have ahigher or lower level of fibrinogen and thus would need an accurateassessment of platelet GPIIb/IIIa receptor blockade to reduce bleedingcomplications due to under assessment of platelet GPIIb/IIIa receptorblockade, or ischemic events due to over assessment of plateletGPIIb/IIIa receptor blockade. In addition, fibrinogen level andfunctionality may change during the trauma of interventional procedures.At this time it is imperative to make an accurate assessment of plateletGPIIb/IIIa receptor blockade in real time, during and following theprocedure.

Thus, there is a need for a method and apparatus for evaluatingcontributors to patient hemostasis both in the presence and absence oftherapies affecting hemostasis such as platelet inhibiting agents,thrombin inhibiting agents and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is graphic illustration representing the mechanism of plateletaggregation.

FIG. 2 is a schematic diagram of a hemostasis analyzer in accordancewith a preferred embodiment of the invention.

FIG. 3 is a plot illustrating a hemostasis profile generated by thehemostasis analyzer shown in FIG. 2.

FIGS. 4-10 are schematic illustrations depicting platelet activationresponsive to various agonist agents.

FIG. 11 illustrates several hemostasis profiles relating to the plateletactivation described in connection with FIGS. 4-10.

FIG. 12 is a schematic diagram of a hemostasis analyzer in accordancewith an alternate embodiment of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the preferred embodiments of the invention, ahemostasis analyzer, such as the Thrombelastograph® (TEG®) hemostasisanalyzer available from Haemoscope Corp., Skokie, Ill., is utilized tomeasure continuously in real time, the hemostasis process from theinitial fibrin formation, through platelet-fibrin GPIIb/IIIa bonding andlysis. While several specific anti-platelet agents are discussed hereinin connection with the preferred embodiments of the invention, it willbe appreciated the invention has application in connection withvirtually any anti-platelet agents. Moreover, it will be furtherappreciated that the invention has application for measuring theefficacy of coagulation enhancing or platelet activating agents.

In accordance with the preferred embodiments of the invention,utilization of the hemostasis analyzer in accordance with the inventiveprotocol permits: confirmation of the attainment of therapeutic level ofGPIIb/IIIa receptor blockade; individualized dosing assessment toevaluate attainment of adequate GPIIb/IIIa receptor blockade;individualized dosing assessment required to reach adequate GPIIb/IIIareceptor blockade; illustration of the rate of diminishment of plateletinhibition or inhibition recovery after treatment withplatelet-inhibition drugs; evaluation of the interaction effect of acombination of thrombolytic and platelet-inhibiting agents, on patienthemostasis.

The present invention may use a hemostasis analyzer 10, such as theThrombelastograph® (TEG®) hemostasis analyzer referenced above, tomeasure the clot's physical properties. An exemplary hemostasis analyzer10 is described in detail in the aforementioned U.S. Pat. No. 6,225,126,and a complete discussion is not repeated here. With reference to FIG.2, to assist in the understanding of the invention, however, a briefdescription of the hemostasis analyzer 10 is provided. The hemostasisanalyzer uses a special stationary cylindrical cup 12 that holds a bloodsample 13. The cup 12 is coupled to a drive mechanism that causes thecup to oscillate through an angle θ, preferably about 4°45′. Eachrotation cycle lasts 10 seconds. A pin 14 is suspended in the bloodsample 13 by a torsion wire 15, and the pin 14 is monitored for motion.The torque of the rotating cup 12 is transmitted to the immersed pin 14only after fibrin-platelet bonding has linked the cup 12 and pin 14together. The strength of these fibrin-platelet bonds affects themagnitude of the pin motion, such that strong clots move the pin 14directly in phase with the cup motion. Thus, the magnitude of the outputis directly related to the strength of the formed clot. As the clotretracts or lyses, these bonds are broken and the transfer of cup motionis diminished.

The rotational movement of the pin 14 is converted by a transducer 16 toan electrical signal, which can be monitored by a computer (not shown inFIG. 2) including a processor and a control program.

The computer is operable on the electrical signal to create a hemostasisprofile corresponding to the measured clotting process. Additionally,the computer may include a visual display or be coupled to a printer toprovide a visual representation of the hemostasis profile. Such aconfiguration of the computer is well within the skills of one havingordinary skill in the art.

As will also be described, based upon an assessment of the hemostasisprofile, the computer, through its control program, may be adapted toprovide dosing recommendations. As shown in FIG. 3), the resultinghemostasis profile 20 is a measure of the time it takes for the firstfibrin strand to be formed, the kinetics of clot formation, the strengthof the clot (measured in millimeters (mm) and converted to shearelasticity units of dyn/cm²) and dissolution of clot. Table I, below,provides definitions for several of these measured parameters.

TABLE I R R time is the period of time of latency from the time that theblood was placed in the TEG ® analyzer until the initial fibrinformation. α a measures the rapidity of fibrin build-up andcross-linking (clot strengthening) MA MA, or Maximum Amplitude in mm, isa direct function of the maximum dynamic properties of fibrin andplatelet bonding via GPIIb/IIIa and represents the ultimate strength ofthe fibrin clot. LY30 LY30 measures the rate of amplitude reduction 30minutes after MA and represents clot retraction, or lysis.

Clinically, these measurements provide a vehicle for monitoringanti-coagulation therapy (e.g. heparin or warfarin), thrombolytictherapy (e.g. tPA, streptokinase, urokinase), effect ofantifibrinolytics (e.g. 8-amino-caproic acid (Amicar®), trasylol(aprotinin), tranexamic acid (TX)), effect of anti-platelet agents (e.g.abciximab (ReoPro®), eptifibatide (Integrilin®), tirofiban (Aggrastat®),blood component transfusion therapy, thrombotic risk assessment incancer and infection, high risk surgery and other conditions which couldpossibly lead to excessive clotting (hypercoagulable conditions) orexcessive bleeding (hypocoagulable conditions). In accordance with theinvention then, the hemostasis analyzer 10 is useful in testing theclinical efficacy of drug therapy to stop fibrinolysis, or the efficacyof thrombolytic drugs to monitor thrombolysis, efficacy of ante plateletagents to monitor platelet inhibition, ischemic or bleedingcomplications.

Quantitatively, the hemostasis analyzer 10 and associated computer plotthe strength of the clot against time, where the onset of clotformation, the reaction time (R), is noted (FIG. 3). This plot alsoindicates the maximum clot strength (or rigidity), MA, of a bloodsample. MA is an overall estimate of platelet-fibrin GPIIb/IIIa bonding,which is used, for example, to guide post-operative blood platelet orfibrinogen replacement therapy. Between platelets and fibrin alone, anabnormally low MA implies that there is an abnormality in bloodplatelets (i.e., a quantitative or functional defect) and/or anabnormality in fibrinogen content in the blood. However, by keepingfibrinogen level and platelet number constant, any change in MA wouldreflect changes in platelet function. Therefore, by testing the sameblood sample two ways, one with an anti-platelet agent and one without,the difference between the two MAs reflects the effect of theanti-platelet agent on platelet function. Similarly, isolating plateletcontribution allows a determination of fibrinogen content, i.e.,available functional fibrinogen.

Platelets play a critical role in mediating ischemic complicationsresulting in stroke and myocardial infarction. Inhibition of plateletfunction by anti-platelet agents (platelet-blocker drugs) such asaspirin, the antibody fragment c7E3 Fab, abciximab (ReoPro®), orclopidogrel, (Plavix®), can result in a dramatic reduction in the riskof death, myocardial infarction, or reocclusion after percutaneoustransluminal coronary angioplasty (PTCA) or intra-arterial thrombolytictherapy (IATT). Administration of excessive amounts of anti-plateletagents could lead to life-threatening bleeding. Therefore, a preciseestimate of platelet function inhibition in a given patient is veryimportant for the monitoring of the drug therapy because of the narrowrisk/therapeutic ratio with this class of drugs.

Using the above strategy, which keeps fibrinogen level and plateletnumber constant, it is possible to properly administer and monitoranti-platelet agents or modify their dosages, or to measure thecontribution of fibrin to MA (MA_(FIB)) and by subtraction to measurethe pure contribution of platelets to MA (MA_(P)) as MA_(P)=MA−MA_(FIB).

Therefore, in accordance with the preferred embodiments of theinvention, to properly monitor anti-platelet agents, the followingprocedure is followed:

-   -   1. The hemostasis analyzer 10, as it is commonly used, measures        platelet function (MA or MA_(P)) that is stimulated by thrombin,        a potent platelet activator that directly activates the        GPIIb/IIIa receptor site. To sensitize MA or MA_(P) to a small        inhibition of platelet function, platelet function should be        activated by a less potent platelet activator than thrombin,        such as ADP that indirectly activates the (GPIIb/IIIa receptor        site. Therefore, when running blood samples in the hemostasis        analyzer 10 in this instance, formation of thrombin is inhibited        with, for example, sodium citrate, heparin, herudin, etc., and        ADP is used instead to activate the platelet function.    -   2. Unfortunately, thrombin is also involved in activating        fibrinogen to fibrin conversion. Having inhibited thrombin        formation in step 1, it is necessary to use another enzyme to        activate fibrinogen. Reptilase, whose sole function is to        activate the fibrinogen to fibrin conversion, is a suitable        enzyme. The clot is now stimulated by reptilase (fibrinogen        activation) and ADP (platelet activation). The strength of the        clot is measured by MA, and the contribution of platelet        function to the strength of the clot is measured by MA_(P), as        described above.    -   3. The clot that is formed by a fibrinogen activator like        reptilase and a platelet activator like ADP is typically weaker        than one developed by thrombin. Therefore, the torsion wire 15        described above may be selected to be sensitive to a weaker clot        and to be able to measure the changes in MA and MA_(P) due to        the small effect of anti-platelet agents such as ReoPro®.        Alternatively, activated Factor XIII (Factor XIIIa) may be        added. Factor XIIIa causes a modification of the fibrin-platelet        bonding from hydrogen bonding to stronger covalent bonding,        enhancing clot strength.

Based on the above, the following protocol may be implemented:

-   -   1. Torsion wire modification of the hemostasis analyzer 10 as        necessary: by producing different strength torsion wires for        various sensitivities to shear force to adequately measuring the        effects of anti-platelet agents of various potencies may be        measured. The sensitivity of the torsion wire is generally        related to its gauge. For increased sensitivity torsion wires        having gauges to sense clot sensitivity in a range from about        150 to about 1000 dyn/cm² are suitable for adaptation to the        hemostasis analyzer described in the aforementioned U.S. Pat.        No. 6,225,236.    -   2. Reptilase-triggered agonist-activated blood sample:        Batroxabin (reptilase, Pentapharm) would be used (15 μl of        reconstituted reptilase reagent) and pre-added to the cup 12 to        activate fibrinogen to fibrin. In addition to the Batroxabin,        ADP (20 μM final concentration) would be pre-added to the cup 12        along with 10 μl of Factor XIIIa. 340 μl of thrombin inhibited        (e.g., citrated, heparinized, etc.) whole blood would be added        to the prewarmed cup 12 containing Batroxabin, ADP and Factor        XIIIa, and maximal clot strength would be assessed. In addition,        a control sample, resulting in complete inhibition of the        platelet contribution the clot strength (MA_(FIB)), would also        be performed with Batroxabin and an anti-platelet agent being        added to the cup 12, providing a measure of the contribution of        fibrin in the absence of the augmenting effect of platelets to        clot strength.

MA_(PB) is measured before the patient is treated with the anti-plateletagent and MA_(PA) is measured after treatment. Platelet inhibition dueto the drug effect will be computed as follows:MA _(PB) =MA _(B) −MA _(FIB)MA _(PA) =MA _(A) −MA _(FIB)Drug inhibition=MA _(PB) −MA _(PA)

It will be appreciated by those having skill in the art that measuringclot strength as described above may require a torsion wire that issensitive to the typically weaker clot formed under conditions ofthrombin inhibition. However, different testing protocols may look toclots having strengths in ranges equal to or greater than typicalthrombin supported clots. In such cases the torsion wire 15 will beselected to be sensitive to such stronger clots. Torsion wires ofseveral gauges providing a range of sensitivities from about 100 dyn/cm²to 25,000 dyn/cm² therefore may be utilized.

It should be further appreciated that the invention has application tomeasuring other parameters of clot formation. For example, thehemostasis analyzer 10 measures the blood clotting process from the timeof test initiation until the initial fibrin formation, through clot ratestrengthening, and clot lysis. Therefore, in accordance in theinvention, it is possible to measure the effect of the presence ofheparin by evaluating the R parameter, which as described aboveindicates the inhibition in initial fibrin formation. It is alsopossible to measure the efficacy of drug therapy on thrombolyticactivity by observing the parameter LY30, which indicates the rate ofclot lysis.

Thrombin, ADP and TxA2 are platelet agonists that initiate the processof platelet aggregation. Through a protease activated receptor (PAR)receptor-mediated response, these agonists cause the expression ofGPIIb/IIIa receptors. The following is a discussion of several factorsand considerations related to this process.

It is well-documented that there is considerable person-to-personvariability in the number of GPIIb/IIIa receptors per platelet and itsligand binding function. Furthermore, variable inhibition of GPIIb/IIIafunction, in part due to the differences in platelet count, may occurafter administration of a fixed, weight-adjusted dose of a plateletblocker. Higher risk patient subgroups, such as diabetic patientsundergoing percutaneous coronary angioplasty (PTCA), may require greaterdoses of platelet inhibition than is currently being attained afterweight-adjusted platelet blocker therapy, which at this time is notindividualized to assure the attainment of adequate GPIIb/IIIa receptorblockade. The potential for hemorrhagic or ischemic events suggests theneed for individualized assessment and projecting of needed dosing toassure the attainment of a therapeutic level of receptor blockade, inreal time. The apparatus and method in accordance with the preferredembodiments of the invention provides this capability.

In contrast to direct platelet GPIIb/IIIa receptor inhibition agents.Plavix® (clopidogrel) is a platelet adenosine diphosphate (ADP) receptorantagonist, inhibiting a class of ADP receptors mediating activation ofplatelet GPIIb/IIIa receptors. Plavix® is taken orally, usually in theform of a loading dose of four 75 mg tablets followed by long-termtherapy of one 75 mg tablet per day prior to and after PTCA or forpatients at high risk for ischemic events. The Plavix® algorithmdictates the same dosing regardless of patient weight or hemostaticprofile. Consequently, treatment with Plavix® can result in increasedbleeding or lack of attainment of an adequate therapeutic level ofplatelet inhibition. Therefore, there is a need to prescribe and monitorindividualized dosing of both platelet GPIIb/IIIa and ADP receptorinhibition (PI) agents.

Thromboxane A₂ (TxA₂) activates the Thromboxane A₂ receptor. OnceThromboxane A₂ receptors are activated, they mediate the activation ofGPIIb/IIIa receptors. Cyclooxygenase is the enzyme necessary in theproduction of Thromboxane A₂, and is inhibited by non-steroidalanti-inflammatory drugs (NSAID).

The result of the activated coagulation protein is the fibrin strandwhich, together with activated platelets at GPIIb/IIIa, formsfibrin(ogin) platelet bonding to produce the final clot. Therefore forplatelet fibrin(ogin) bonding to occur or to take place plateletGPIIb/IIIa receptors have to be activated. Therefore platelet agonistsare constructed toward activation of the GPIIb/IIIa receptor directly,as by thrombin, or indirectly as by ADP and Thromboxane A₂. Consequentlyplatelet inhibitor drugs are specifically targeted towards inhibitingthese agonists as illustrate in FIGS. 4-10.

With reference to FIGS. 4-10, A platelet 30 has a GPIIb/IIIa receptorsite 32, an ADP receptor site 34 and a TxA₂ receptor site 36. As shownin FIG. 4, addition of ADP agonist activates the ADP receptor site 34(illustrated by arrow 38), which activates the (GPIIb/IIIa receptor site(illustrated by arrow 40). A platelet adenosine diphosphate (ADP)receptor antagonist, such as Plavix®, inhibits the ADP receptor site 34(phantom arrow 42 in FIG. 5), and thus the GPIIb/IIIa site is notactivated in response to the presence of the ADP agonist (phantom arrow44). Therefore, in the presence of an ADP receptor antagonist, an ADPagonist only activates platelets that are not inhibited. The result is areduction in clot strength, illustrated as the tracing MA_(pi) in FIG.11 as compared to the clot strength with complete platelet activationillustrated as the tracing MA_(kh) in FIG. 11.

ReoPro®, Integrilin®, and Aggrastat® inhibit the GPIIb/IIIa receptor 32directly. When the ADP agonist is added, it activates the ADP receptor34 (arrow 46 in FIG. 6) but is stopped at the GPIIb/IIIa receptor(phantom arrow 48). Therefore, in the presence of GPIIb/IIIa inhibitors,only the non-inhibited platelets will be activated by the ADP agonistresulting in correspondingly reduced clot strength, e.g., MA_(pi)illustrated in FIG. 11.

Thromboxane A₂ activates the platelet TxA₂ receptor 36 (arrow 50 in FIG.7), which in turn activates the GPIIb/IIIa receptor 32 (arrow 52).Arachidonic acid (AA) is a precursor to Thromboxane A₂ and is convertedto Thromboxane A₂ in the presence of Cyclooxygenase. Non-steroidalanti-inflammatory drugs (NSAID) inhibits Cyclooxygenase (phantom arrow54 in FIG. 8), and thus the GPIIb/IIIa site is also not activated in thepresence of a TxA₂ agonist (phantom arrow 56) resulting in acorresponding reduction in clot strength, e.g., MA_(pi) in FIG. 11.Therefore an AA platelet agonist can only activate the GPIIb/IIIa site32 when NSAID is not taken.

Thrombin is the enzyme that cleaves soluble fibrinogen into fibrinstrands. It is also the most potent platelet activator, strongly anddirectly increasing the expression and activation of platelet GPIIb/IIIareceptors (arrow 58 in FIG. 9). ReoPro®, Integrilin®, and Aggrastat®agents inhibit GPIIb/IIIa receptors responsive to ADP or TxA₂ agonists;however, thrombin will still result in GPIIb/IIIa activation (arrow 60in FIG. 10). Certain hemostasis assays, such as the above-referencedTEG® assay, may be arranged to produce thrombin in the process of clotformation. The increased expression and strong activation of plateletGPIIb/IIIa by thrombin overcomes the GPIIb/IIIa inhibitors to provide afull platelet activation clot strength (MA_(kh) in FIG. 11). In vivo,thrombin escaping into the circulation is immediately inhibited byendogenous antithrombin agents, localizing the hemostatic response tothe site of injury. Ex vivo, for example when blood is placed in atesting apparatus, thrombin generation is continuous throughout the clotformation process. Due to the absence of entothelium, inhibitory,activity is limited to the endogenous antithrombin agents in the sampleand remains viable to cleave fibrinogen and mediate activation ofGPIIb/IIIa receptors. Thus, as described herein, thrombin is suppressedto allow isolation and evaluation of other hemostasis factors andtherapies.

With the foregoing discussion, it is therefore possible to furtherspecify a protocol for monitoring platelet inhibitors, such asGPIIb/IIIa, ADP and Thromboxane A₂ platelet inhibitors. Referring toFIG. 12, an apparatus 10′ may include a plurality of hemostasis analysisdevices, such as the hemostasis analyzer 10 shown in FIG. 2, and tourare shown as devices 10 a, 10 b, 10 c and 10 d, to respectively testcorresponding blood samples 13 a, 13 b, 13 c and 13 d.

A first sample 13 a of heparinized whole blood is prepared and loadedinto the first hemostasis analyzer 10 a. A fibrin activator 17, such asa combination of Reptilase and Factor XIIIa, is added to the sample cup12. The activator 17 may be pre-added to the sample cup 12 or the cup 12may be treated with the activator 17. Alternatively, the activator 17may be added to the blood sample 13 a after it is added to the cup 12.About 10 μl of activator 17 is added to a 340 μl blood sample. The assayis completed and the resulting clot strength is measured. This clotstrength may be referred to as MA_(f), as it represents only thecontribution of fibrin with substantially no platelet activation in viewof the absence of any platelet agonist.

A second sample 13 b of heparinized whole blood is prepared and isloaded into the second hemostasis analyzer 10 b. It should be noted thatthe a single cell hemostasis analyzer may be used serially for each ofthe assays, more than one single cell analyzer may be used, ormulti-cell analyzers may be used. For example, the TEG® hemostasisanalyzer in standard configuration has two testing cells, which mayoperate simultaneously. Two TEG® hemostasis analyzers may be used toperform each of the four assays according to this exemplary protocol orone TEG® hemostasis analyzer may be used. Moreover, the two TEG®hemostasis analyzers may be networked, making in essence a single fourcell testing device.

For the second sample 13 b, the activator 17 is added to the sample, andan ADP agonist 18 is also added in appropriate proportion to the secondsample 13 b. For example, 20 μM of ADP agonist may be added to a 340 μlsecond sample of heparinized whole blood. The assay is completed and theresulting clot strength is measured. This clot strength may be referredto as MA_(pi1), as it represents the contributions of fibrin andplatelets uninhibited by any administered ADP platelet inhibitionagents.

A third sample 13 c of heparinized whole blood is prepared and is loadedinto a third hemostasis analyzer 10 c. The fibrin activator 17 added tosample 13 c, and a Thromboxane A₂ agonist 19 is added in appropriateproportion to the third sample 13 c. For example, 10 μl of ArachidonicAcid (AA) may be added to a 340 μl third sample of heparinized wholeblood. The assay is completed and the resulting clot strength ismeasured. This clot strength may be referred to as MA_(pi2) as itrepresents the contributions of fibrin and platelets uninhibited byadministered TxA₂ platelet inhibition agents.

A fourth sample 13 d of heparinized whole blood is prepared and isloaded into a fourth hemostasis analyzer 10 d. For the fourth sample 13d, heparinase 21 and kaolin 22 are used to neutralize the effect of theheparin in the fourth sample 13 d. The assay is completed and theresulting clot strength is measured. This clot strength may be referredto as MA_(kh), and it measures the maximum MA with platelet activationdue to the use of heparinase and kaolin to neutralize the heparin in thesample 13 d and to enable the production of thrombin which activates theGPIIb/IIIa receptors.

The value MA_(pi)−MA_(f) measures the unique contributions of theuninhibited platelets by PI agents where platelet inhibition can be byadministration of Reopro®, Aggrastat®, Integrilin®, ADP and NSAIDs. Apercentage reduction in MA due to platelet inhibition is then calculatedfor each of MA_(pi1) and MA_(pi2) according the equation:Percent Platelet Activation=[(MA_(pij)−MA_(f))/MA_(kh)−MA_(f))]*100,where the value MA_(kh)−MA_(f) measures the unique contributions of thefully activated platelets. Thus, the medical practitioner may observethe effect of platelet inhibition therapy, isolated into componenteffects, and make dosing recommendations accordingly. Alternatively,percent platelet inhibition may be determined as100−[(MA_(pij)−MA_(f))/MA_(kh)−MA_(f))]*100.

It will be appreciated that the sample cups 12 in the foregoing assaysmay be prepared in advance to contain the appropriate agents inaccordance with the foregoing described protocol. In that regard, thesample cups may be color coded to identify the particular agentscontained within the cup. Sets of sample cups 12 may be packaged tofacilitate the assays. Still further, the agent materials. e.g. theactivator 17, may be distributed in color coded vials for ease ofidentification and for loading into the sample cups 12 either before orafter the corresponding blood sample is loaded into the sample cup.

Assays in accordance with various preferred embodiments of theinvention, such as the examples described above, may employ thrombinsuppression, via in vitro or otherwise introduction of a suitablethrombin inhibitor, such as heparin. However, with thrombin suppressionand a corresponding absence of thrombin contribution to hemostasis, e.g.cleavage of fibrinogen to form fibrin, additional reagents are requiredto compensate for the lack of thrombin and its contributing factorsbeyond platelet activation.

Human platelets are activated by thrombin cleavage of PAR-1 and/or PAR-4causing platelet aggregation and secretion. Antibody blockade of bothPAR-1 and PAR-4 prevents virtually all thrombin-mediated plateletaggregation and secretion. This suggests that PAR-1 and PAR-4 mediatemost, if not all, of the thrombin mediated signaling in human platelets.With PAR-1/PAR-4 inactivation and the thrombin's active site stillintact, thrombin's pro-coagulant actions, e.g., cleavage of fibrinogento form fibrin, activation of the serine protease FXI (Factor XI) andtransglutaminase FXIII and other non-enzymatic coagulation factorsremain intact. Thus, in the foregoing protocols, suppression ofthrombin's platelet activating function by anti-body blockade of PAR-1and PAR-4 may be employed. At the same time, additional reagents are notrequired to preserve the pro-coagulant action of thrombin, e.g., cleavefibrinogen to fibrin and to activate Factor XIII to Factor XIIIa forfibrin cross-linking and the like.

The invention has been described in terms of several preferredembodiments. One of skill in the art will appreciate that the inventionmay be otherwise embodied without departing from its fair scope, whichis set forth in the subjoined claims.

1. A method of evaluating patient hemostasis, the method comprising:testing a first portion of a blood sample obtained from a patient todetermine a first quantitative indication of a first blood samplehemostasis characteristic, the first portion first being prepared sothat during testing of the first portion the first blood samplehemostasis characteristic is distinguishable from other blood samplehemostasis characteristics; testing a second portion of the blood sampleobtained from the patient to determine a second quantitative indicationof a second blood sample hemostasis characteristic, the second portionfirst being prepared so that during testing of the second portion thesecond blood sample hemostasis characteristic is distinguishable fromother blood sample hemostasis characteristics; calculating a third bloodsample hemostasis characteristic using the first blood sample hemostasischaracteristic and the second blood sample hemostasis characteristic;and evaluating a hemostasis condition of the patient from which theblood sample was taken at least based upon the third blood samplehemostasis characteristic.
 2. The method of claim 1, wherein the thirdblood sample characteristic is indicative of blood platelet contributionto hemostasis.
 3. The method of claim 1, wherein the third blood samplecharacteristic is indicative of fibrin contribution to hemostasis. 4.The method of claim 1, wherein the third blood sample characteristic isindicative of fibrinogen content in the blood sample.
 5. The method ofclaim 1, wherein the blood sample is prepared in vitro to suppressthrombin activation; the first portion is first prepared in vitro toactivate fibrinogen to fibrin conversion and the second portion is firstprepared in vitro to activate fibrinogen to fibrin conversion and toprovide platelet activation.
 6. The method of claim 5, wherein the bloodsample is prepared in vitro to suppress thrombin activation by additionof a protease activated receptor (PAR) antibody blockade affective forPAR-1 or PAR-4 receptor blockade.
 7. The method of claim 1, wherein theblood sample reflects in vivo administration of hemostasis therapy tothe patient, the first portion is first prepared in vitro to neutralizethe hemostasis therapy and the second portion is first prepared in vitroto activate fibrinogen to fibrin conversion and to provide plateletactivation.
 8. The method of claim 7, wherein the hemostasis therapycomprises administration to the patient of a platelet inhibitiontherapy, administration to the patient of an anti-coagulation therapy,administration to the patient of an antifibrinolytics therapy oradministration to the patient of a blood component transfusion therapy.9. The method of claim 1, wherein each of the first quantitativeindication and the second quantitative indication comprises respectiveblood clot strength indications.
 10. The method of claim 1, wherein eachof the first quantitative indication and the second quantitativeindication comprises respective time to initial blood clot formationindications.
 11. The method of claim 1, wherein each of the firstquantitative indication and the second quantitative indication comprisesrespective rate of blood clot formation indications.
 12. The method ofclaim 1, wherein each of the first quantitative indication and thesecond quantitative indication comprises respective time to blood clotlysis indications.
 13. The method of claim 1, wherein the first bloodhemostasis characteristic represents a fibrin contribution to hemostasisand the second blood hemostasis characteristic represents an activatedplatelets in the presence of an in vivo hemostasis therapy contributionto hemostasis.
 14. The method of claim 1, comprising testing anadditional portion of the blood sample obtained from the patient todetermine an additional quantitative indication of an additional bloodsample hemostasis characteristic, the additional portion first beingprepared so that during testing of the additional portion the additionalblood sample hemostasis characteristic is distinguishable from otherblood sample hemostasis characteristics and wherein the additional bloodsample hemostasis characteristic represents a contribution to hemostasisof substantially complete platelet activation, and wherein determining athird blood sample hemostasis characteristic comprises calculating thethird blood sample hemostasis characteristic using the first bloodsample hemostasis characteristic, the second blood sample hemostasischaracteristic and the additional blood sample characteristic.
 15. Themethod of claim 1, wherein the first portion is first prepared using afibrin activator.
 16. The method of claim 15, wherein the fibrinactivator comprises reptilase, Factor XIIIA and combinations thereof.17. The method of claim 1, wherein the second portion is first preparedusing an adenosine diphosphate (ADP) site activator.
 18. The method ofclaim 17, wherein the ADP site activator comprises an ADP agonist. 19.The method of claim 1, wherein the second portion is first preparedusing a Thromboxane A₂ site activator.
 20. The method of claim 19,wherein the Thromboxane A₂ site activator comprises arachidonic acid.21. The method of claim 1, wherein the first portion is prepared using aprotease activated receptor (PAR)-1 and PAR-4 inhibitor.
 22. The methodof claim 1, comprising substantially simultaneously testing the firstportion and the second portion.
 23. The method of claim 1, comprisingproviding a first hemostasis testing cell and a second hemostasistesting cell, and simultaneously testing the first portion in the firsthemostasis testing cell and the second portion in the second hemostasistesting cell.
 24. The method of claim 23, wherein the first and secondhemostasis testing cells comprise first and second testing cells ofsingle hemostasis analyzer.